Recombinant (poly)peptides and proteins for applications in basic research, diagnostics, and therapy, such as antibody molecules, vaccines, hormones, and growth factors, are produced using a wide variety of genetically engineered organisms that include both prokaryotic and eukaryotic cells. However, the vast majority of recombinant peptides or proteins include post-translational modifications that cannot be mimicked or re-produced when using prokaryotic host cells. For this reason, mammalian gene expression systems have turned out to represent a preferred choice.
Mammalian expression systems based on Chinese Hamster ovary (CHO) cells are widely used in production of recombinant protein. Apart from lymphoid cell lines, CHO cells represent one of the few cell types allowing for simple and efficient high-density suspension batch culture of animal cells. Furthermore, the use of CHO cells results in high product yields, while lymphoid cells are more difficult to culture at an industrial scale. In view of considerable costs for recombinant production of polypeptides and proteins, it is also of utmost importance to maximize the yield of recombinant protein per bioreactor run. Process parameters that have considerable impact on product yield include inter alia the cell culture conditions, the copy number of the nucleic acids (genes) to be expressed, the efficiency with which these genes are transcribed and the corresponding mRNAs are translated, the stability of the mRNA, and the like.
Accordingly, improvements of the strength or transcriptional activity of the regulatory genetic elements controlling gene expression constitute a particularly critical factor in order to augment the yield of recombinant protein produced. Even small incremental increases in transcriptional activity at the single cell level will finally translate into considerable improvements in product yield in high-density industry-scale batch cultures.
The vast majority of mammalian gene expression systems employ expression vectors encoding the heterologous nucleic acid sequences to be expressed under the control of promoter regulatory sequences derived from viruses. Two of the most frequently used viral regulatory elements in these expression cassettes are those of the human cytomegalovirus (hCMV) immediate early genes 1 and 2 (IE1 and IE2). However, a disadvantage associated with the use of hCMV IE1 and IE2 regulatory elements is their pronounced species specificity.
U.S. Pat. No. 5,866,359 discloses that gene expression from such hCMV promoter can be improved by co-expressing adenoviral EIA protein under the control of a weak promoter. EIA is a multifunctional transcription factor which may act on cell cycle regulation and has both independent transcriptional activating and repressing functional domains. Fine tuning of EIA expression is crucial to achieve the ideal balance between gene transactivation and any negative impact on cell cycle progression. However, overexpression of EIA expression could reduce the capacity of the cell to synthesize the recombinant protein of interest.
U.S. Pat. No. 5,591,639 describes vectors comprising, upstream (5′) of a heterologous nucleic acid sequence to be expressed, the enhancer, promoter, and complete 5′-untranslated region of the major immediate early gene of the human cytomegalovirus (hCMV-MIE) including intron A (i.e. the first natural intron). However, if the first 400 bp (5′-end) of this sequence (total length of about 2100 bp) were present, poor gene expression rates were observed in both COS7 and CHO cells (Chapman, B. S. et al. (1991) Nucl. Acids Res. 19, 3979-3986).
The transcriptional activity of the regulatory elements of the immediate early genes of the murine cytomegalovirus (mCMV) is higher than that of the hCMV counterparts without exhibiting the pronounced species preference observed for the human sequences (Addison, C. L. et al. (1987) J. Gen. Virol. 78, 1653-1661).
However, attempts to enhance the activity of the mCMV IE promoter regulatory elements, analogously to the hCMV counterparts, by insertion of the natural first intron of the murine major immediate early gene downstream (3′) of the mCMV IE promoter failed (cf. inter alia EP patent 1 525 320 81). However, the generation of expression vectors comprising a chimeric cassette composed of the regulatory elements of mCMV IE1 and the natural first intron of the human major immediate early gene resulted in product yields comparable to the use of the fully human sequences (cf., e.g., WO 2006/111387 A2). Similar gene expression rates were also obtained for expression vectors comprising the mCMV IE2 regulatory sequences (cf. inter alia EP patent 1 601 776 81).
Thus, there still remains a need for improved mammalian gene expression systems resulting in high yields of the recombinant polypeptides or proteins produced. In particular, there is a need for mammalian gene expression systems that overcome the above-mentioned limitations, that is, expression systems based on the mCMV or the hCMV promoter sequences but achieving higher expression rates (and thus, product yields) than with the available system
Accordingly, it is an object of the present invention to provide such gene expression systems, primarily suitable expression constructs and corresponding mammalian host cells.